Attention is directed to an article by the inventors and a colleague, entitled "An Amber Suppressor tRNA Gene Derived By Site-Specific Mutagenesis: Cloning and Function in Mammalian Cells" in the Proceedings of the National Academy of Science Vol. 79, No. 19, pp. 5813-5817 (1982), and an article entitled "Establishment of Mammalian Cell Lines Containing Multiple Nonsense Mutations and Functional Suppressor tRNA Genes" in Cell Vol. 31, No. 1, pp. 137-146 (1982), both herein incorporated by reference.
Of the sixty-four possible codon triplets which form the genetic code, there are three nonsense codons which are known to stop protein production at cellular ribosomes; the other sixty-one triplets in the code correspond to one or another amino acid. When genetic instruction are translated at the ribosomes, the amino acids are strung together to form complex polypeptides. However, when a nonsense codon is read, it is interpreted as a stop signal terminating the protein production. The three nonsense codons are UAG (amber), UAA (ochre) and UGA (opal).
Nonsense codons are sometimes caused by mutations and, as a result, genetic phenotypes may not be expressed. Despite the presence of the gene directing expression, a crucial protein may not be produced because of an unwanted stop signal reaches a ribosome and terminates the unfinished protein. There are also times when one wants to manipulate a gene to include a nonsense codon. In either case, there exists a need for a suppression mechanism which would permit the cellular ribosomes to "read through" such stop signals when they are unwanted.
Suppressor genes for prokaryotic cells, such as E. coli bacteria, have been isolated and studied. See, for example, Goldberg, Biological Regulation and Development Vol. 1 Gene Expression, pp 433-475 (1979). Such suppressors operate by producing tRNA molecules which possess an altered anticodon for reading the nonsense codon as if it were a normal instruction. For example, an amber mutation results in the creation of a UAG codon in messenger RNA. An amber suppressor gene produces tRNA with a CUA anticodon, thereby inserting an amino acid at the UAG site and permitting continued translation.
While suppressor genes have been characterized for E. coli bacteria and similar work is being conducted on yeasts, the isolation of suppressor genes in mammalian cells has not been successful due to the complexity of such cells and the lack of adequate selection protocols. Moreover, the transformation of bacterial suppressor genes into mammalian cells has not been successful because of the nature of higher organism cells. We are not aware of any successfully synthesized suppressor genes for mammalian cells, other than the ones reported herein. Temple et al in an article entitled "Construction of a Functional Human Suppressor tRNA gene: an approach to gene therapy for Bthalassaemia" Vol. 296 Nature pp 537-542 (Apr., 1982) report the synthesis of a suppressor gene and its expression in frog eggs.
There exists a need for suppressor genes for mammalian cells. The availability of mammalian cell lines with well characterized tRNA suppressor mutants would greatly facilitate genetic analysis of nonsense mutants in mammalian cells and in particular animal viral genomes. Moreover, the availability of such cell lines should open new pathways for the use of mammalian host cells as growth media for vaccines and other valuable products.